The aim of this project is to assess the effects of aging and caloric restriction (CR) at a cellular and biochemical level of analysis, to identify physiological mechanisms associated with these effects, and to evaluate nutritional interventions that might alter age-related declines in function. Interventions include various nutritional, hormonal and pharmacological treatments. CR affects metabolic regulation to induce an overall phenotypic change leading to a decrease in cellular proliferation and growth rates. CR induces measurable changes on circulating levels of several hormones and growth factors that regulate cell growth and proliferation. Serum obtained from CR animals alters growth, proliferation and stress responses of cells in culture. We have demonstrated that it is possible to investigate certain aspects of CR using this in vitro approach. This approach lends itself to a more rapid investigation of possible mechanisms and, perhaps more importantly to the research, development and rapid evaluation of interventions that would be able to induce or promote a phenotype similar to that seen with CR, essentially a CR mimetic. In response to damage or stress, cells attempt to repair and defend themselves, but if unsuccessful, they often undergo programmed cell death, or apoptosis. Numerous studies show that aging is associated with increased rates of stress-induced apoptosis, and the cumulative effects of cell loss have been implicated in various diseases including neurodegeneration, retinal degeneration, cardiovascular disease, and frailty. In mammals, CR delays the onset of numerous age-associated diseases including cancer, atherosclerosis, and diabetes, and can greatly increase lifespan. The molecular mechanisms underlying this effect are not known. In yeast, CR extends lifespan by increasing the activity of the Sir2 protein, a member of the nicotinamide adenine dinucleotide (NAD+)?dependent deacetylases family. In lower organisms, lifespan can be extended by the presence of extra copies of the SIR2/Sir-2.1 gene, or by a group of small-molecules that activate the sirtuins. In mammals, it is becoming increasingly apparent that SIRT1 is a key regulator of cell defenses and survival in response to stress. A major cause of aging is thought to result from the cumulative effects of cell loss over time. We have recently shown that expression of mammalian Sir2 (SIRT1) is induced in CR rats as well as in human cells that are treated with serum from these animals. Insulin and insulin-like growth factor 1 (IGF-1) attenuated this response. SIRT1 inhibits, through K70, Bax mediated stress-induced apoptotic cell death. Thus, CR could extend lifespan by inducing SIRT1 expression and promoting the long-term survival of irreplaceable cells. CR extends lifespan in a variety of animal model systems and reduces oxidative stress during aging. At least in part, the reduction in oxidative stress may be explained by the fact that animals on CR reach a new bioenergetic equilibrium. The major component in the bioenergetic pathway is the mitochondria electron transport chain but also the plasma membrane (PM) redox system (PMRS). Ubiquinone is the central molecule of the PMRS and protects the membrane under different stress conditions. Aging induces general macromolecular damage that can be prevented and reversed by CR. Preliminary data suggest that several components of the PMRS are altered during aging and that several of these changes are modified by CR in rats and mice. Analysis of the bioenergetic balance between mitochondria and PM in rats and mice on CR can provide the information that might explain the enhanced resistance to oxidative stress that CR affords during aging. It has been proposed that life span is inversely related to the degree of membrane phospholipid unsaturation and that elucidation of this relationship can provide insight on the mechanism for lifespan extension with CR. Such hypotheses that link lipids to aging solely through modulation of membrane susceptibility to peroxidation, however, may be too simplistic since they ignore the effect lipid alterations have on other membrane-associated processes. Such processes include proton (and other ion) leaks, ROS production, apoptosis, and maintenance of antioxidant systems. Membrane-induced alterations in any of these processes could have major influences on oxidative stress and life span. We have investigated the effects of CR on a few of these membrane processes, and shown that CR stimulates the plasma membrane antioxidant system, and attenuates apoptosis. The mechanisms responsible for these CR-induced alterations in membrane functions are unknown; however, the recent development of high throughput analytical methods could make this investigation more feasible. These techniques may be used to quantify lipids, such as cardiolipin and ceramide, that have functions which could contribute to the actions of CR in addition to identifying other lipids that may be altered by CR. Considering the central role that membranes play in regulating oxidative stress and apoptosis, we hypothesize that CR causes a rapid and sustained alteration in plasma and mitochondrial membrane composition that results in a new bioenergetic balance leading to decreases in both ROS production and membrane oxidative damage. We are currently pursuing a number of specific knock-out models systems, such as NRF-2 and NQO-1 mice, to confirm this hypothesis.